Value of lipocalin 2 as a potential biomarker for bacterial meningitis.

OBJECTIVES: Central nervous system (CNS) infections are common causes of morbidity and mortality worldwide. We aimed to discover protein biomarkers that could rapidly and accurately identify the likely cause of the infections, essential for clinical management and improving outcome. METHODS: We applied liquid chromatography tandem mass spectrometry on 45 cerebrospinal fluid (CSF) samples from a cohort of adults with and without CNS infections to discover potential diagnostic biomarkers. We then validated the diagnostic performance of a selected biomarker candidate in an independent cohort of 364 consecutively treated adults with CNS infections admitted to a referral hospital in Vietnam. RESULTS: In the discovery cohort, we identified lipocalin 2 (LCN2) as a potential biomarker of bacterial meningitis (BM) other than tuberculous meningitis. The analysis of the validation cohort showed that LCN2 could discriminate BM from other CNS infections (including tuberculous meningitis, cryptococcal meningitis and virus/antibody-mediated encephalitis), with sensitivity of 0.88 (95% confident interval (CI), 0.77-0.94), specificity of 0.91 (95% CI, 0.88-0.94) and diagnostic odds ratio of 73.8 (95% CI, 31.8-171.4). LCN2 outperformed other CSF markers (leukocytes, glucose, protein and lactate) commonly used in routine care worldwide. The combination of LCN2, CSF leukocytes, glucose, protein and lactate resulted in the highest diagnostic performance for BM (area under the receiver operating characteristics curve, 0.96; 95% CI, 0.93-0.99). Data are available via ProteomeXchange with identifier PXD020510. CONCLUSIONS: LCN2 is a sensitive and specific biomarker for discriminating BM from a broad spectrum of other CNS infections. A prospective study is needed to assess the diagnostic utility of LCN2 in the diagnosis and management of CNS infections.

Supramolecular attack particles are autonomous killing entities released from cytotoxic T cells.

Cytotoxic T lymphocytes (CTLs) kill infected and cancerous cells. We detected transfer of cytotoxic multiprotein complexes, called supramolecular attack particles (SMAPs), from CTLs to target cells. SMAPs were rapidly released from CTLs and were autonomously cytotoxic. Mass spectrometry, immunochemical analysis, and CRISPR editing identified a carboxyl-terminal fragment of thrombospondin-1 as an unexpected SMAP component that contributed to target killing. Direct stochastic optical reconstruction microscopy resolved a cytotoxic core surrounded by a thrombospondin-1 shell of ~120 nanometer diameter. Cryo-soft x-ray tomography analysis revealed that SMAPs had a carbon-dense shell and were stored in multicore granules. We propose that SMAPs are autonomous extracellular killing entities that deliver cytotoxic cargo targeted by the specificity of shell components.

Somatic gene editing ameliorates skeletal and cardiac muscle failure in pig and human models of Duchenne muscular dystrophy.

Frameshift mutations in the DMD gene, encoding dystrophin, cause Duchenne muscular dystrophy (DMD), leading to terminal muscle and heart failure in patients. Somatic gene editing by sequence-specific nucleases offers new options for restoring the DMD reading frame, resulting in expression of a shortened but largely functional dystrophin protein. Here, we validated this approach in a pig model of DMD lacking exon 52 of DMD (DMDΔ52), as well as in a corresponding patient-derived induced pluripotent stem cell model. In DMDΔ52 pigs1, intramuscular injection of adeno-associated viral vectors of serotype 9 carrying an intein-split Cas9 (ref. 2) and a pair of guide RNAs targeting sequences flanking exon 51 (AAV9-Cas9-gE51) induced expression of a shortened dystrophin (DMDΔ51-52) and improved skeletal muscle function. Moreover, systemic application of AAV9-Cas9-gE51 led to widespread dystrophin expression in muscle, including diaphragm and heart, prolonging survival and reducing arrhythmogenic vulnerability. Similarly, in induced pluripotent stem cell-derived myoblasts and cardiomyocytes of a patient lacking DMDΔ52, AAV6-Cas9-g51-mediated excision of exon 51 restored dystrophin expression and amelioreate skeletal myotube formation as well as abnormal cardiomyocyte Ca2+ handling and arrhythmogenic susceptibility. The ability of Cas9-mediated exon excision to improve DMD pathology in these translational models paves the way for new treatment approaches in patients with this devastating disease.

Colorectal cancer liver metastatic growth depends on PAD4-driven citrullination of the extracellular matrix.

Citrullination of proteins, a post-translational conversion of arginine residues to citrulline, is recognized in rheumatoid arthritis, but largely undocumented in cancer. Here we show that citrullination of the extracellular matrix by cancer cell derived peptidylarginine deiminase 4 (PAD4) is essential for the growth of liver metastases from colorectal cancer (CRC). Using proteomics, we demonstrate that liver metastases exhibit higher levels of citrullination and PAD4 than unaffected liver, primary CRC or adjacent colonic mucosa. Functional significance for citrullination in metastatic growth is evident in murine models where inhibition of citrullination substantially reduces liver metastatic burden. Additionally, citrullination of a key matrix component collagen type I promotes greater adhesion and decreased migration of CRC cells along with increased expression of characteristic epithelial markers, suggesting a role for citrullination in promoting mesenchymal-to-epithelial transition and liver metastasis. Overall, our study reveals the potential for PAD4-dependant citrullination to drive the progression of CRC liver metastasis.

Debridement and implant retention in the management of hip periprosthetic joint infection: outcomes following guided and rapid treatment at a single centre.

AIMS: To analyse the effectiveness of debridement and implant retention (DAIR) in patients with hip periprosthetic joint infection (PJI) and the relationship to patient characteristics. The outcome was evaluated in hips with confirmed PJI and a follow-up of not less than two years. PATIENTS AND METHODS: Patients in whom DAIR was performed were identified from our hip arthroplasty register (between 2004 and 2013). Adherence to criteria for DAIR was assessed according to a previously published algorithm. RESULTS: DAIR was performed as part of a curative procedure in 46 hips in 42 patients. The mean age was 73.2 years (44.6 to 87.7), including 20 women and 22 men. In 34 hips in 32 patients (73.9%), PJI was confirmed. In 12 hips, the criteria for PJI were not fulfilled and antibiotics stopped. In 41 (89.1%) of all hips and in 32 (94.1%) of the confirmed PJIs, all criteria for DAIR were fulfilled. In patients with exogenous PJI, DAIR was performed not more than three days after referral. In haematogenous infections, the duration of symptoms did not exceed 21 days. In 28 hips, a single debridement and in six hips two surgical debridements were required. In 28 (87.5%) of 32 patients, the total treatment duration was three months. Failure was noted in three hips (9%). Long-term follow-up results (mean 4.0 years, 1.4 to 10) were available in 30 of 34 (88.2%) confirmed PJIs. The overall successful outcome rate was 91% in 34 hips, and 90% in 30 hips with long-term follow-up results. CONCLUSION: Prompt surgical treatment with DAIR, following strict diagnostic and therapeutic criteria, in patients with suspected periprosthetic joint infection, can lead to high rates of success in eradicating the infection. Cite this article: Bone Joint J 2017;99-B:330-6.

Using an Integrated -Omics Approach to Identify Key Cellular Processes That Are Disturbed in the Kidney After Brain Death.

In an era where we are becoming more reliant on vulnerable kidneys for transplantation from older donors, there is an urgent need to understand how brain death leads to kidney dysfunction and, hence, how this can be prevented. Using a rodent model of hemorrhagic stroke and next-generation proteomic and metabolomic technologies, we aimed to delineate which key cellular processes are perturbed in the kidney after brain death. Pathway analysis of the proteomic signature of kidneys from brain-dead donors revealed large-scale changes in mitochondrial proteins that were associated with altered mitochondrial activity and morphological evidence of mitochondrial injury. We identified an increase in a number of glycolytic proteins and lactate production, suggesting a shift toward anaerobic metabolism. Higher amounts of succinate were found in the brain death group, in conjunction with increased markers of oxidative stress. We characterized the responsiveness of hypoxia inducible factors and found this correlated with post-brain death mean arterial pressures. Brain death leads to metabolic disturbances in the kidney and alterations in mitochondrial function and reactive oxygen species generation. This metabolic disturbance and alteration in mitochondrial function may lead to further cellular injury. Conditioning the brain-dead organ donor by altering metabolism could be a novel approach to ameliorate this brain death-induced kidney injury.

Hypoxic regulation of RIOK3 is a major mechanism for cancer cell invasion and metastasis.

Hypoxia is a common feature of locally advanced breast cancers that is associated with increased metastasis and poorer survival. Stabilisation of hypoxia-inducible factor-1α (HIF1α) in tumours causes transcriptional changes in numerous genes that function at distinct stages of the metastatic cascade. We demonstrate that expression of RIOK3 (RIght Open reading frame kinase 3) was increased during hypoxic exposure in an HIF1α-dependent manner. RIOK3 was localised to distinct cytoplasmic aggregates in normoxic cells and underwent redistribution to the leading edge of the cell in hypoxia with a corresponding change in the organisation of the actin cytoskeleton. Depletion of RIOK3 expression caused MDA-MB-231 to become elongated and this morphological change was due to a loss of protraction at the trailing edge of the cell. This phenotypic change resulted in reduced cell migration in two-dimensional cultures and inhibition of cell invasion through three-dimensional extracellular matrix. Proteomic analysis identified interactions of RIOK3 with actin and several actin-binding factors including tropomyosins (TPM3 and TPM4) and tropomodulin 3. Depletion of RIOK3 in cells resulted in fewer and less organised actin filaments. Analysis of these filaments showed reduced association of TPM3, particularly during hypoxia, suggesting that RIOK3 regulates actin filament specialisation. RIOK3 depletion reduced the dissemination of MDA-MB-231 cells in both a zebrafish model of systemic metastasis and a mouse model of pulmonary metastasis. These findings demonstrate that RIOK3 is necessary for maintaining actin cytoskeletal organisation required for migration and invasion, biological processes that are necessary for hypoxia-driven metastasis.

Cofactor Strap regulates oxidative phosphorylation and mitochondrial p53 activity through ATP synthase.

Metabolic reprogramming is a hallmark of cancer cells. Strap (stress-responsive activator of p300) is a novel TPR motif OB-fold protein that contributes to p53 transcriptional activation. We show here that, in addition to its established transcriptional role, Strap is localised at mitochondria where one of its key interaction partners is ATP synthase. Significantly, the interaction between Strap and ATP synthase downregulates mitochondrial ATP production. Under glucose-limiting conditions, cancer cells are sensitised by mitochondrial Strap to apoptosis, which is rescued by supplementing cells with an extracellular source of ATP. Furthermore, Strap augments the apoptotic effects of mitochondrial p53. These findings define Strap as a dual regulator of cellular reprogramming: first as a nuclear transcription cofactor and second in the direct regulation of mitochondrial respiration.

The treatment and outcome of peri-prosthetic infection of the ankle: a single cohort-centre experience of 34 cases.

The treatment of peri-prosthetic joint infection (PJI) of the ankle is not standardised. It is not clear whether an algorithm developed for hip and knee PJI can be used in the management of PJI of the ankle. We evaluated the outcome, at two or more years post-operatively, in 34 patients with PJI of the ankle, identified from a cohort of 511 patients who had undergone total ankle replacement. Their median age was 62.1 years (53.3 to 68.2), and 20 patients were women. Infection was exogenous in 28 (82.4%) and haematogenous in six (17.6%); 19 (55.9%) were acute infections and 15 (44.1%) chronic. Staphylococci were the cause of 24 infections (70.6%). Surgery with retention of one or both components was undertaken in 21 patients (61.8%), both components were replaced in ten (29.4%), and arthrodesis was undertaken in three (8.8%). An infection-free outcome with satisfactory function of the ankle was obtained in 23 patients (67.6%). The best rate of cure followed the exchange of both components (9/10, 90%). In the 21 patients in whom one or both components were retained, four had a relapse of the same infecting organism and three had an infection with another organism. Hence the rate of cure was 66.7% (14 of 21). In these 21 patients, we compared the treatment given to an algorithm developed for the treatment of PJI of the knee and hip. In 17 (80.9%) patients, treatment was not according to the algorithm. Most (11 of 17) had only one criterion against retention of one or both components. In all, ten of 11 patients with severe soft-tissue compromise as a single criterion had a relapse-free survival. We propose that the treatment concept for PJI of the ankle requires adaptation of the grading of quality of the soft tissues.

A regulatory circuit that involves HR23B and HDAC6 governs the biological response to HDAC inhibitors.

Histone deacetylase (HDAC) is an emergent anticancer target, and HR23B is a biomarker for response to HDAC inhibitors. We show here that HR23B has impacts on two documented effects of HDAC inhibitors; HDAC inhibitors cause apoptosis in cells expressing high levels of HR23B, whereas in cells with low level expression, HDAC inhibitor treatment is frequently associated with autophagy. The mechanism responsible involves the interaction of HDAC6 with HR23B, which downregulates HR23B and thereby reduces the level of ubiquitinated substrates targeted to the proteasome, ultimately desensitising cells to apoptosis. Significantly, the ability of HDAC6 to downregulate HR23B occurs independently of its deacetylase activity. An analysis of the HDAC6 interactome identified HSP90 as a key effector of HDAC6 on HR23B levels. Our results define a regulatory mechanism that involves the interplay between HR23B and HDAC6 that influences the biological outcome of HDAC inhibitor treatment.

Association between granulomatous colitis in French Bulldogs and invasive Escherichia coli and response to fluoroquinolone antimicrobials.

BACKGROUND: French Bulldogs develop a form of granulomatous colitis (GC) with histopathological resemblance to GC of Boxer dogs (GCB). GCB is associated with mucosally invasive Escherichia coli whose eradication correlates with clinical remission. HYPOTHESIS/OBJECTIVES: To characterize the clinical and histopathological features, presence or absence of invasive colonic bacteria, and response to fluoroquinolones in French Bulldogs with GC. ANIMALS: A total of 6 French Bulldogs with a histological diagnosis of GC. METHODS: Retrospective study of medical records. Bacterial colonization was evaluated using 16S rRNA probes for eubacteria and E. coli. Biopsy specimens from 3 dogs were cultured for bacteria. Clinical response to fluoroquinolone antimicrobials was determined. RESULTS: All dogs were ≤1 year of age with hematochezia that was refractory to empirical therapy. Clinicopathologic and fecal analysis did not reveal abnormalities. Abdominal ultrasound revealed patchy thickening of the colon in 4/5 dogs and regional lymphadenopathy in 5/5. Colonoscopic abnormalities included irregularly thickened and ulcerated mucosa, hyperemia, and overt bleeding in 4/6 cases. Multifocal accumulations of PAS-positive macrophages and intramucosal E. coli were present in colonic biopsies of all 6 dogs. Administration of enrofloxacin (5/6) or marbofloxacin (1/6) at 4.4-10 mg/kg (median 10 mg/kg) PO q24h for 6-10 weeks was associated with clinical improvement within 5-14 days. All dogs remained in remission over a 3-30 month follow-up period. CONCLUSIONS: Granulomatous colitis in young French Bulldogs is associated with the presence of invasive E. coli and closely parallels GCB. Treatment with fluoroquinolone antimicrobials can induce lasting clinical remission.

Release of pamidronate from poly(ethyleneimine)/cellulose sulphate complex nanoparticle films: an in situ ATR-FTIR study.

In situ ATR-FTIR spectroscopy was used as a screening method to quantify the relative release of pamidronate (PAM) from films of polyelectrolyte (PEL) complex (PEC) particles. Stable colloid PEC particles consisting of poly(ethyleneimine) (PEI) and cellulose sulphate (CS) loaded with PAM were obtained by PEL complexation featuring hydrodynamic radii between 60 and 90 nm and a cationic or anionic surface charge dependent on the mixing ratio n-/n+=0.9 or 1.1, respectively. Respective bare unloaded PEC particles showed smaller hydrodynamic radii. PAM loaded PEC particles were casted from dispersion onto Ge model substrates and dried forming stable films in contact to water. By in situ ATR-FTIR spectroscopy it could be shown, that PAM/PEC particle films contacting to water resulted in a time dependent retarded release of PAM from the PEC matrix, while PAM from a pure drug film was immediately released. Cationic PAM loaded PEC particles of PEI/CS showed smaller initial burst and long term release compared to anionic one at similar PAM/PEI ratios. With increasing PAM/PEI ratio the initial burst could be minimized to around 30% and the residual long term amount of PAM optimized to 50% for PAM/PEC samples casted from 0.002 M dispersions. A further improvement of the release performance was achieved either by prerinsing the dry film in H(2)O or by rising the PEC concentration from 0.002 M to 0.01 M revealing an initial burst of around 5% and long term residual PAM content of around 75%. ATR-FTIR and TRANS-FTIR analysis of the PAM release from equivalent PEC samples revealed similar kinetic courses and parameters justifying the use of the Ritger/Peppas two parameter model. Applying this model PAM/PEC samples casted from 0.002 M dispersions revealed exponent values of b≪0.5 suggesting PAM dissolution in the PEC matrix, while for those casted from 0.01 M b values close to 0.5 were obtained suggesting hindered dissolution and diffusion. A model describing different retention modalities of PAM in PEC particle is suggested.

Sequential targeting of CFTR by BAC vectors generates a novel pig model of cystic fibrosis.

Cystic fibrosis (CF) is the most common lethal inherited disease in Caucasians and is caused by mutations in the CFTR gene. The disease is incurable and medical treatment is limited to the amelioration of symptoms or secondary complications. A comprehensive understanding of the disease mechanisms and the development of novel treatment options require appropriate animal models. Existing CF mouse models fail to reflect important aspects of human CF. We thus generated a CF pig model by inactivating the CFTR gene in primary porcine cells by sequential targeting using modified bacterial artificial chromosome vectors. These cells were then used to generate homozygous CFTR mutant piglets by somatic cell nuclear transfer. The homozygous CFTR mutants lack CFTR protein expression and display severe malformations in the intestine, respiratory tract, pancreas, liver, gallbladder, and male reproductive tract. These phenotypic abnormalities closely resemble both the human CF pathology as well as alterations observed in a recently published CF pig model which was generated by a different gene targeting strategy. Our new CF pig model underlines the value of the CFTR-deficient pig for gaining new insight into the disease mechanisms of CF and for the development and evaluation of new therapeutic strategies. This model will furthermore increase the availability of CF pigs to the scientific community.

OS045. Multi-dimensional protein identification technology analysis of syncytiotrophoblast vesicles released from perfused preeclampsia placentas.

INTRODUCTION: In pre-eclampsia, the consequences of poor placentation lead to the second stage of pre-eclampsia, which involves activation of a maternal systemic inflammatory response (MSIR). Endothelial and other inflammatory cellular dysfunction cause the diverse features which characterise the disorder. We have previously shown that syncytiotrophoblast microvesicles (STBM) are pro-inflammatory and circulate in increased amounts in pre-eclamptic women. We hypothesise that multiple placental "danger signals" are carried by STBM into the maternal circulation in increased amounts in PE with pro-inflammatory, anti-angiogenic and pro-coagulant activity, implicating STBM in the pathophysiology of PE. OBJECTIVES: To characterise the proteins carried by STBM from normal and PE placentas. For the first time multi-dimensional protein identification technology (MudPIT) was used to derive the proteome profiles of normal and PE placenta STBM. METHODS: STBM were prepared from placentas (normal term: n=9 and PE: n=5) by dual lobe perfusion, isolated by ultracentrifugation and stored at -80°C. Normal and PE derived placenta STBM pools were then subjected to MudPIT analysis. RESULTS: 538 proteins unique to PE STBM, 604 proteins unique to normal STBM and 1421 proteins common to both preparations were found. Preliminary analysis indicates the presence of alarmins (HSP70, and galectin 3), exosomal proteins (CD63,CD9,CD81), immunoregulatory molecules (CD26,CD200,CD47,Galectin 1), complement and complement regulatory molecules (C1q,C3,CD55,CD59 and vitronectin), amino acid transporters (CD98) and anti-angiogenic molecules (endoglin). Our analysis also reveals that proteins known to be elevated in blood before, or at, the time of pre-eclampsia are elevated or unique in STBM from PE placentas, including Fetuin A, Inter-alpha (globulin) inhibitor H4, Serum amyloid P component, Apolipoprotein H (or B2GP1) and Apolipoprotein AII. Thus, as predicted, a large number of circulating molecules are associated with STBM. The inter-relationships between proteins that are unique to either PE or normal pregnancy and the processes in which they are involved are being determined by Ingenuity Pathways Analysis software. In terms of biofunctions, preliminary analysis shows that proteins unique to PE STBM have a highly significant association (p<10(-11)) with 6 disease pathways including inflammatory, immunological, cardiovascular and reproductive system diseases and organ injury, whereas for proteins unique to normal STBM only protein synthesis was significant at the same level. CONCLUSION: STBM contain a heterogeneous population of vesicles that convey a large repertoire of placental proteins into the maternal circulation. The profound differences between PE and normal STBM indicate their pro-inflammatory potential.

Label-free quantitative proteomics reveals differentially regulated proteins influencing urolithiasis.

Urinary proteins have been implicated as inhibitors of kidney stone formation (urolithiasis). As a proximal fluid, prefiltered by the kidneys, urine is an attractive biofluid for proteomic analysis in urologic conditions. However, it is necessary to correct for variations in urinary concentration. In our study, individual urine samples were normalized for this variation by using a total protein to creatinine ratio. Pooled urine samples were compared in two independent experiments. Differences between the urinary proteome of stone formers and nonstone-forming controls were characterized and quantified using label-free nano-ultraperformance liquid chromatography high/low collision energy switching analysis. There were 1063 proteins identified, of which 367 were unique to the stone former groups, 408 proteins were unique to the control pools, and 288 proteins were identified for comparative quantification. Proteins found to be unique in stone-formers were involved in carbohydrate metabolism pathways and associated with disease states. Thirty-four proteins demonstrated a consistent >twofold change between stone formers and controls. For ceruloplasmin, one of the proteins was shown to be more than twofold up-regulated in the stone-former pools, this observation was validated in individuals by enzyme-linked immunosorbent assay. Moreover, in vitro crystallization assays demonstrated ceruloplasmin had a dose-dependent increase on calcium oxalate crystal formation. Taken together, these results may suggest a functional role for ceruloplasmin in urolithiasis.

Age-related loss of orexin/hypocretin neurons.

Aging is associated with many physiological alterations-such as changes in sleep patterns, metabolism and food intake-suggestive of hypothalamic dysfunction, but the effects of senescence on specific hypothalamic nuclei and neuronal groups that mediate these alterations is unclear. The lateral hypothalamus and contiguous perifornical area (LH/PFA) contains several populations of neurons, including those that express the neuropeptides orexin (hypocretin) or melanin-concentrating hormone (MCH). Collectively, orexin and MCH neurons influence many integrative homeostatic processes related to wakefulness and energy balance. Here, we determined the effect of aging on numbers of orexin and MCH neurons in young adult (3-4 months) and old (26-28 months) Fisher 344/Brown Norway F1 hybrid rats. Aged rats exhibited a loss of greater than 40% of orexin-immunoreactive neurons in both the medial and lateral (relative to the fornix) sectors of the LH/PFA. MCH-immunoreactive neurons were also lost in aged rats, primarily in the medial LH/PFA. Neuronal loss in this area was not global as no change in cells immunoreactive for the pan-neuronal marker, NeuN, was observed in aged rats. Combined with other reports of altered receptor expression or behavioral responses to exogenously-administered neuropeptide, these data suggest that compromised orexin (and, perhaps, MCH) function is an important mediator of age-related homeostatic disturbances of hypothalamic origin. The orexin system may represent a crucial substrate linking homeostatic and cognitive dysfunction in aging, as well as a novel therapeutic target for pharmacological or genetic restoration approaches to preventing or ameliorating these disturbances.

Cell-mediated transgenesis in rabbits: chimeric and nuclear transfer animals.

The ability to perform precise genetic engineering such as gene targeting in rabbits would benefit biomedical research by enabling, for example, the generation of genetically defined rabbit models of human diseases. This has so far not been possible because of the lack of functional rabbit embryonic stem cells and the high fetal and perinatal mortality associated with rabbit somatic cell nuclear transfer. We examined cultured pluripotent and multipotent cells for their ability to support the production of viable animals. Rabbit putative embryonic stem (ES) cells were derived and shown capable of in vitro and in vivo pluripotent differentiation. We report the first live born ES-derived rabbit chimera. Rabbit mesenchymal stem cells (MSCs) were derived from bone marrow, and multipotent differentiation was demonstrated in vitro. Nuclear transfer was carried out with both cell types, and embryo development was assessed in vitro and in vivo. Rabbit MSCs were markedly more successful than ES cells as nuclear donors. MSCs were transfected with fluorescent reporter gene constructs and assessed for nuclear transfer competence. Transfected MSCs supported development with similar efficiency as normal MSCs and resulted in the first live cloned rabbits from genetically manipulated MSCs. Reactivation of fluorescence reporter gene expression in reconstructed embryos was investigated as a means of identifying viable embryos in vitro but was not a reliable predictor. We also examined serial nuclear transfer as a means of rescuing dead animals.

Dichroic ATR-FTIR spectroscopy on oriented α-helical poly(l-lysine) multilayered with polyanions.

The preparation and spectroscopic and microscopic characterization of oriented polyelectrolyte multilayers (PEM) interesting for defined nanostructured functional materials and surfaces are reviewed. Oriented PEM were generated by consecutively adsorbing α-helical poly(l-lysine) (PLL) and oppositely charged polyanions like poly(vinylsulfate) (PVS) or poly(styrene sulfonate) (PSS) at silicon substrates texturized by parallel nanoscopic surface grooves, respectively. Dichroic Attenuated Total Reflexion Fourier Transform Infrared (ATR-FTIR) spectroscopy was used to study the conformation and macromolecular order of stiff polyelectrolytes within PEM. High order parameters up to S=0.82 (S=1 for high, S=0 for low order) were obtained from the dichroic ratios of the Amide I and Amide II bands suggesting a significant alignment of charged α-helical polypeptides in PEM. For PEM consisting of PLL/polyanion the S values significantly increased with increasing molecular weight of PLL and with decreasing molecular weight of the polyanion. These spectroscopic findings were supported by SFM images on PEM-PLL/PVS with high molecular PLL and PEM-PLL/PSS with low molecular PSS, which both showed anisotropically oriented worm-like structures, while PEM-PLL/PVS with low molecular PLL and PEM-PLL/PSS with high molecular PSS showed no orientation features.

Tunable superconducting phase transition in metal-decorated graphene sheets.

We have produced graphene sheets decorated with a nonpercolating network of nanoscale tin clusters. These metal clusters both efficiently dope the graphene substrate and induce long-range superconducting correlations. We find that despite structural inhomogeneity on mesoscopic length scales (10-100 nm), this material behaves electronically as a homogenous dirty superconductor with a field-effect tuned Berezinskii-Kosterlitz-Thouless transition. Our facile self-assembly method establishes graphene as an ideal tunable substrate for studying induced two-dimensional electronic systems at fixed disorder and our technique can readily be extended to other order parameters such as magnetism.